The long-term goal of this project is to understand the fundamental molecular mechanisms of cardioprotection Our central hypothesis is that inducible nitric oxide synthase (iNOS) and heme oxygenase-1 (HO-1) protect the heart from ischemia-reperfusion (I-R)injury by partially inhibiting mitochondrial electron transport, which decreases the generation of free radicals and prevents mitochondrial permeability transition (MPT). We propose that these changes prevent ischemic cell death and are responsible for the cardioprotective effects of the late phase of preconditioning (PC). We will use electron paramagnetic resonance measurements to quantify the generation of oxygen-derived free radicals and nitric oxide and determine whether free radicals generation and oxidative stress are attenuated in preconditioned hearts subjected to I-R (Aim 1). To test the antioxidant role of iNOS and HO-1, we will measure free radical generation and oxidative stress in hearts transduced with recombinant adeno-associated virus (rAAV) carrying the iNOS or HO-1 gene. Conversely, we will determine whether I-R-induced oxidative stress and radical generation are exacerbated in the hearts of iNOS or HO-1 null mice (Aim 2). To examine whether the cardioprotective effects of late PC are derived from inhibition of MPT, we will measure permeability transition and the activity of respiratory complexes in mitochondria isolated from naive and preconditioned hearts and test whether PC decreases I-R-induced MPT in isolated perfused hearts (Aim 3). To determine the role of iNOS and HO-1, we will examine whether inhibition or the lack of these proteins abolishes the effects of PC on MPT and conversely whether hearts of mice transduced with rAAV/iNOS or rAAV/HO-1 are resistant to I-R-induced MPT. We will determine whether iNOS and HO-1 prevent mitochondrial injury by associating with the mitochondria and whether localization of these proteins to the mitochondria (by using mitochondria-specific leader sequence) is sufficient to confer cardioprotection (Aim 4).